Although there is much interest in the clinical use of mesenchymal stem cells for repair and regeneration of skeletal tissues, the molecular mechanisms controlling differentiation are not well understood. Differentiation of mesenchymal cells into chondrocytes requires the coordinated expression of specific regulatory genes, of which few have been characterized. Zfp28, a candidate chondrogenic regulator, is a Cys2His2 zinc finger protein (ZFP) having N-terminal KRAB-A and B domains. Most KRAB zinc finger proteins have been shown to function as transcriptional repressors. Zfp28 is expressed in a limited subset of adult tissues, and is expressed in mouse embryos at the onset of skeletogenesis. Zfp28 is expressed in the chondrogenic murine ATDC5 cell line in a differentiation stage-specific manner. Overexpression of Zfp28 results in nuclear translocation, downregulation of Col2a1 mRNA, and inhibition of chondrogenic differentiation. Coexpression of Zfp28 results in downregulation of Col2a1 promoter activity in ATDC5 cells. The Zfp28 gene resides on mouse chromosome 7 (Mm7) within a cluster of imprinted zinc-finger genes, some of which are expressed in limb development. ZFP28, the human ortholog of Zfp28 is on 19q13.4, in a region having synteny with the region of Mm7 containing Zfp28. As zinc finger proteins have been shown to be highly divergent between species, conservation of the Zfp28 sequence between human and mouse suggests an important function. We hypothesize that Zfp28 functions as a transcriptional repressor of genes expressed during chondrogenesis. Specific aims to test this hypothesis are: (1) To determine whether Zfp28 is required for chondrogenesis in vivo. We will produce Zfp28 conditional knockout mice to determine the relationship of this gene to skeletal morphogenesis. The skeletal phenotype of the embryos will be analyzed for gross morphology, histology and gene expression. (2) to examine the role of Zfp28 in chondrogenic differentiation in vitro. We will analyze chondrogenic differentiation following manipulation of Zfp28 expression in ATDC5 cells or in the conditionally immortalized mesenchymal progenitor cell line BMC9. We will study the effect of Zfp28 expression upon transcription from collagen II and XI, and aggrecan promoter-reporter constructs. We will identify Zfp28 target genes by coupled chromatin immunoprecipitation and mouse promoter microarray analysis. This project will result in a better understanding of the molecular events regulating the differentiation of mesenchymal stem cells toward chondrocytes. This knowledge may accellerate progress toward novel therapies to repair or regenerate damaged or osteoarthritic cartilage. [unreadable] [unreadable] [unreadable]